RAMAN FLOW CYTOMETRY FOR DRUG DISCOVERY AND DIAGNOSIS

用于药物发现和诊断的拉曼流式细胞术

• 批准号: 8169398
• 负责人: JOHN P NOLAN
• 金额: $ 3.34万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169398
• 关键词: Academia; Affinity; Area; Binding; Biomedical Engineering; Biomedical Research; Cells; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Detection; Diagnosis; Diagnostic; Engineering; Event; Flow Cytometry; Fluorescence; Funding; Government; Grant; Individual; Industry; Information Systems; Institution; Label; Libraries; Measurement; Microscopy; Microspheres; Molecular; Molecular Analysis; Optics; Peptide Library; Peptides; Performance; Plant Resins; Polymers; Property; Proteins; Raman Spectrum Analysis; Reporter; Reporting; Research; Research Personnel; Research Project Grants; Resources; Route; Science; Screening procedure; Sorting - Cell Movement; Source; Speed; System; Techniques; Technology; Testing; Toxin; United States National Institutes of Health; Work; abstracting; base; biological systems; chemical synthesis; drug discovery; high throughput screening; instrument; instrumentation; nanostructured; new technology; novel; particle; pathogen; therapeutic development

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Abstract The National Flow Cytoemtry Resource and the Bioengineering Research Partnership (BRP) to develop Raman Flow Cytometry for Diagnostics and Drug Discovery both aim to develop novel flow instrumentation for biomedical science. The two projects have many areas of synergy where collaborations will be established to speed the progress of both projects. Background The ability to make quantitative, high throughput molecular measurements of biological systems is a critical need for many areas of biomedical research. The Bioengineering Research Partnership (BRP) to develop Raman Flow Cytometry for Diagnostics and Drug Discovery aims to develop a powerful new analytical platform for high throughput screening and selection based on Raman Flow Cytometry. This Partnership will develop new analytical instrumentation, optically encoded polymer resins for chemical synthesis and screening, and nanostructured materials with unique optically properties for sensitive reporting and encoding. The new technology will perform Raman spectroscopy on single particles in flow to enable new applications in sensitive multiplexed detection, drug discovery, and diagnostics. The Raman Flow Cytometry instrumentation and applications will be developed by a Partnership involving engineers, biologists, and chemists from academia, government and industry. We have modified a commercial particle sorter (the COPAS) to detect individual Raman vibrational bands from single particles and sorted these particles based on their optical signature. We are also developing the ability to collect and analyze complete Raman spectra from single particles (1). In parallel, the Partnership has developed new encoding and reporting strategies for multiplexed molecular analysis and separation. This Raman Flow Cytometry technology will be applied to the development of therapeutics and diagnostics for bacterial pathogens and their toxins. Raman Flow Cytometry will be an important and general new analytical and separation capability that will impact many areas of basic and applied biomedical research. Approach The BRP discussed above will develop new Raman analysis capabilities for flow cytometry. This BRP will collaborate on all of the research projects of the NFCR. First, as sensitivity is critical to Raman analysis, the NFCR will work with the BRP to provide acoustically focused flow cells for high sensitivity measurements without a concurrent loss of particle analysis rate. Second, Peptide libraries can be synthesized to specifically bind a number of different proteins. Dr. Nolan's Bioengineering Research Partnership is developing technologies to rapidly select peptides that bind toxin proteins. The BRP is synthesizing many peptide libraries on large particles (>50 microns) that will bind fluorescent protein targets. The approach planned by the BRP has been to provide Raman analysis of the microspheres concurrently with fluorescent reporter binding via flow cytometry analysis, which will allow high speed decoding of the compound on the Raman microspheres via its Raman barcode for microspheres that are positive for binding events (2, 3). Provision of large particle sorting technology to this project, will aid its progression in two ways: sorted particles could be re-analyzed to confirm the online flow analysis and high speed sorting of the rare particles that bind the fluorescent reporters followed by established Raman microscopy technologies [2, 3] to decode the Raman signature that identifies the compound on the microsphere could provide an alternate route selection of peptides synthesized on Raman microsphere libraries. We will use these libraries as demonstration approach to identify fluorescent toxin binders to peptide bearing microspheres. The identity of the peptide will be identified via mass-spec of sorted microspheres or by integral Raman signatures within the microsphere identified via Raman microscopy. Finally, the BRP and the NFCR are developing spectral instrumentation for orthogonal purposes and with different approaches. The NFCR and the BRP will collaborate on many technical aspects of spectral flow cytometry. Specifically, he NFCR will work with the BRP on spectral flow cytometry, data systems, parallel analysis and large particle sorting. We will sort large particle libraries provided by the BRP using fluorescence techniques. In the out years of this proposal we will provide the large particle sorting technology to be implemented with their systems. We will also implement ORCA on the BRP spectral systems and provide them with line driven flow cells to maximize the sensitivity of the BRPs spectral systems. The BRP through Dr. Nolan will provide large particle microsphere libraries and protein targets that can be screened using fluorescence techniques. We will focus on demonstration that we can detect the binding of fluorescently labeled proteins to peptide libraries generated to have affinities for a variety of toxins. Dr. Nolan's lab will serve as a beta testing facility for data systems, line drives and new high speed parallel analysis technologies and sorters and will communicate with the NFCR to optimize instrument performance.

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 摘要 国家流式细胞仪资源和生物工程研究伙伴关系（BRP）开发用于诊断和药物发现的拉曼流式细胞仪，旨在开发用于生物医学科学的新型流式仪器。这两个项目有许多协同作用的领域，将建立合作，以加快两个项目的进展。 背景 对生物系统进行定量、高通量分子测量的能力是生物医学研究的许多领域的关键需求。生物工程研究伙伴关系（BRP）开发用于诊断和药物发现的拉曼流式细胞术，旨在开发一种强大的新分析平台，用于基于拉曼流式细胞术的高通量筛选和选择。该合作伙伴关系将开发新的分析仪器，用于化学合成和筛选的光学编码聚合物树脂，以及具有独特光学特性的纳米结构材料，用于敏感的报告和编码。这项新技术将对流动中的单个颗粒进行拉曼光谱分析，从而在敏感的多重检测、药物发现和诊断方面实现新的应用。拉曼流式细胞仪仪器和应用将由一个合作伙伴关系开发，该合作伙伴关系包括来自学术界、政府和工业界的工程师、生物学家和化学家。我们已经修改了商业粒子分选仪（COPAS）检测单个拉曼振动带从单个粒子和分选这些粒子的基础上，他们的光学签名。我们还在开发收集和分析单个粒子的完整拉曼光谱的能力（1）。与此同时，该伙伴关系为多重分子分析和分离开发了新的编码和报告策略。这种拉曼流式细胞术技术将应用于细菌病原体及其毒素的治疗和诊断的开发。拉曼流式细胞术将是一种重要的和通用的新的分析和分离能力，将影响基础和应用生物医学研究的许多领域。 方法 上面讨论的BRP将开发用于流式细胞术的新的拉曼分析能力。该BRP将在NFCR的所有研究项目上进行合作。首先，由于灵敏度对拉曼分析至关重要，因此NFCR将与BRP一起工作，以提供用于高灵敏度测量的声学聚焦流动池，而不会同时损失颗粒分析速率。第二，可以合成肽文库以特异性结合许多不同的蛋白质。诺兰博士的生物工程研究伙伴关系正在开发技术，以快速选择结合毒素蛋白的肽。BRP正在大颗粒（>50微米）上合成许多肽库，这些肽库将结合荧光蛋白靶标。BRP计划的方法是通过流式细胞术分析同时提供微球的拉曼分析和荧光报告分子结合，这将允许通过其拉曼条形码对拉曼微球上的化合物进行高速解码，用于结合事件呈阳性的微球（2，3）。为该项目提供大颗粒分选技术将从两个方面帮助其进展：可以重新分析分选的颗粒以确认结合荧光报告物的稀有颗粒的在线流动分析和高速分选，随后是已建立的拉曼显微技术[2，3]来解码识别微球上的化合物的拉曼特征可以提供在拉曼微球文库上合成的肽的替代路线选择。我们将使用这些库作为演示方法来鉴定荧光毒素结合剂的肽轴承微球。肽的身份将通过分选微球的质谱或通过拉曼显微镜鉴定的微球内的积分拉曼特征来鉴定。最后，BRP和NFCR正在开发用于正交目的和不同方法的光谱仪器。NFCR和BRP将在光谱流式细胞术的许多技术方面进行合作。 具体来说，他NFCR将与BRP在光谱流式细胞术，数据系统，并行分析和大颗粒分选。我们将使用荧光技术对BRP提供的大颗粒库进行分类。在本提案的最后几年，我们将提供与他们的系统一起实施的大颗粒分选技术。我们还将在BRP光谱系统上实施ORCA，并为其提供线驱动流动池，以最大限度地提高BRP光谱系统的灵敏度。 Nolan博士的BRP将提供大颗粒微球库和可以使用荧光技术筛选的蛋白质靶点。我们将重点展示我们可以检测荧光标记蛋白质与肽库的结合，这些肽库对各种毒素具有亲和力。Nolan博士的实验室将作为数据系统、线驱动器和新的高速并行分析技术和分选机的beta测试设施，并将与NFCR进行沟通，以优化仪器性能。